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Drugs from Emasculated Hormones The Principle of Syntopic Antagonism (Nobel Lecture).

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Drugs from Emasculated Hormones:
The Principle of Syntopic Antagonism (Nobel Lecture) **
By Sir James W. Black*
In this lecture I want to give an outline of the early stages in
the discovery of adrenaline 8-receptor antagonists and of the
histamine H,-receptor antagonists. I will end with a brief
personal view about future research.
Adrenaline b-Receptor Antagonists
The work that is the theme of this lecture began in the
early summer of 1958 when I joined Imperial Chemical Industries’ Pharmaceuticals Division. I had gone there to pursue a very clear project that had been developing in my mind
for several years. The idea had clinical, therapeutic, physiological, and pharmacological elements.
Clinically, angina pectoris was known to be precipitated
by anxiety and emotion just as well as by exercise. Indeed,
the initiation of pain by an injection of adrenaline had been
used as a diagnostic test. Partial thyroidectomy had been
found to relieve severe angina pectoris whether or not associated with hyperthyroidism. At that time, tachycardia seemed
to me to be the connecting link in these disorders.
Therapeutically, nitroglycerine could quickly relieve and
attack of angina. Nitroglycerine also produced facial flush
and headache. The relief of angina was attributed to similar
vasodilatation in the coronary arteries. However, the newer,
synthetic, selective coronary vasodilators, such as dipyridamole, were clinically ineffective despite the enhanced coronary artery dilatation that they provided. Here was a question mark against the widely practiced industrial strategy of
seeking better drugs to increase coronary blood flow for
angina.
Physiologically, Smith and Lawson“] had found that hyperbaric oxygen, at two atmospheres pressure, reduced the
incidence of ventricular fibrillation associated with occlusion
of a coronary artery, even though the oxygen-carrying capacity of the blood had increased by a maximum of only
25 %. Might not an equivalently small decrease in the myocardial demand for oxygen be just as effective? That was my
question.
Myocardial oxygen consumption is determined by the
work of the heart and is a function of arterial blood pressure
and heart rate. Lowering blood pressure by systemic vasodilatation might dangerously reduce the perfusion pressure
and blood flow through disease-narrowed coronary arteries.
Indeed, hypotension was known to be able to induce a heart
attack. Heart rate, on the other hand, is largely determined
by the cardiac autonomic nervous system. Heart rate would
[*] Prof. James Black
James Black Foundation
King’s College School of Medicine and Dentistry
68 Half Moon Lane. Dulwich, GB-London SE24 9 JE (UK)
[**I
886
Copyright 0The Nobel Foundation 1989. We thank the Nobel Foundation, Stockholm, for permission to print this lecture.
Q VCH VerlagsgesellschaJLmhH, 0-6940 Weinhelm, 1989
thus be reduced by cardiac sympathetic blockade. In addition, there was much discussion in those days about a postulated “anoxiating” action of adrenaline, proposing that the
price of rapidly increasing cardiac power was a decrease in
cardiac metabolic efficiency.
These clinical, therapeutic, and physiological features of
hearts coping with coronary artery disease all seemed to
point to the potential advantage of annulling the actions of
the sympathetic hormones, noradrenaline and adrenaline,
on the heart.
Pharmacologically, the antiadrenaline drugs were a wellrecognized class in 1958. All of them showed a pattern of
actions similar to those seen by Dale[21with the ergot alkaloids. Characteristically, they reversed the blood pressure
rise produced by adrenaline to a fall in pressure, but they did
not suppress the associated tachycardia. Konzettr3] had
shown that isoprenaline, the purely synthetic isopropyl derivative of noradrenaline, produced only the actions such as
tachycardia, vasodilatation, and bronchodilatation which
the antiadrenaline drugs were not able to suppress. These
were the actions of isoprenaline that Ahlquisti4] could not
explain on the basis of Cannon and Rosenbleuth’s prevailing
hypothesis involving sympathins E and I.i51 Ahlquist went on
to propose that the wide-spread physiological effects of adrenaline were mediated by two classes of receptors, a and 8. In
this new classification, the antiadrenaline drugs of the day
were a-receptor antagonists and isoprenaline was a selective
stimulant of p-receptors.
So I started at I.C.I. with a clear goal-I wanted to find a
8-receptor antagonist. I expected this to reduce pulse rates at
rest and during exercise and hoped that it would decrease the
susceptibility of patients to angina pectoris. The unknown
factor for me at that time was the significance of adrenaline’s
“anoxiating” activity.
John Stephenson was the medicinal chemist assigned to
work with me. As no compounds were known to annul the
actions of adrenaline on the heart, the program had to be
cold-started. The structure of isoprenaline (Fig. l), the selective fl-receptor stimulant, was our only clue. We thought that
if N-substitution of adrenaline with an isopropyl group produced a selective agonist, then perhaps substitution with a
different, larger group might produce a selective antagonist.
We thought that symmetrical, “doubled-up’’ analogues of
isoprenaline, dibenzylethylamines, might be interesting targets.
We were making compounds and testing them, admittedly
without success, when, early in 1959, we read Powell and
Slaw’s report 161 about the properties of dichloroisoprenaline (DCI), an analogue of isoprenaline in which the ring
hydroxyl groups were replaced by chlorine atoms (Fig. 1). In
trying to exploit the bronchodilator properties of isoprenaline by making a long-acting variant, the Lilly group had
discovered a compound that intrigued them by displaying - instead of isoprenaline’s bronchodilator activity - the
oS70-OR33/R9/0707-0886 $02.50/0
Angeew. Chem. Int. Ed. Engl. 28 (1989) 886-894
adrenaline
pronethalol
isoprenaline
dichloroisoprenaline
b)
diphenethylornine
propranolol
Fig. I . Chemical structures of adrenaltne-related compounds.
opposite property, namely antagonism. Soon afterwards,
Moran and Perkins['] reported that DCI could annul the
inotropic effects of adrenaline on the heart and classified
DCI as a P-receptor antagonist. Stephenson immediately
made some DCI for us to test.
We had started our bioassays using the classical Langendorff preparation, the isolated, spontaneously beating,
guinea-pig heart. Isoprenaline is a powerful stimulant of
both the rate and force of beating in this preparation, but we
measured the amplitude ofcontractions, which compounded
both changes. In this system DCI turned out to be as powerful a stimulant as isoprenaline and so was not at all what we
were looking for. We had also developed a technique for
simultaneously recording blood pressure and heart rate, in
analogue form, in anesthetized animals. Potential antagonists could now be given economically by slow intravenous
infusion, and this allowed the effect on a wide range of systems to be monitored. Here, too, the powerful stimulant
effects of DCI on heart rate were clearly seen, although there
was less hypotension due to vasodilatation than we had expected (Fig. 2). On the basis of these experiments, we decided
that DCI was not the lead we needed.
As analytical pharmacologists, what we are allowed to see
of a new molecule's properties is totally dependent on the
techniques of bioassay we use. The prismatic qualities of an
assay distort our view in obscure ways and degrees. Our only
defense lies in restless improvement in technique and experimental design, in the hope that collimation of several techniques will improve the reliability of our vision. We would
make the change self-consciously today, but then it was intuitive.
We developed a new in vitro assay based on guinea-pig
cardiac papillary muscles as a way of measuring the contractile effects of isoprenaline independently of rate changes.
Then, we reassessed many early compounds, including DCI.
On the new preparation, DCI had no stimulant activities
itself but simply antagonized the effects of adrenaline and
isoprenaline, although the stimulant activity on pacemaker
tissue could be clearly seen in the atrial preparation. We were
astonished. Today, we classify DCI as a partial agonist.
Ariens[81and R . P.Stephenson[91had introduced the concept
A n g w . Chem. I n ( . Ed. Engl. 28 (1989) 886-894
l
S
l
S
l
S
l
S
Fig. 2. Effects of a)DCI (IOOpg kg-' min-') and bjpronethalol (1OOpg
kg-' min-I) infusion upon heart rate and blood pressure responses to injections of isoprenaline (I) (0.4 p g kg-' 1.v.) and lo Sympathetic stimulation (S)
(square wave pulses, 10-ms duration, 2.5 V, 15 pulses per second for 30 s) in
anesthetized cats. Lower axis shows time markers in one-minute intervals.
of partial agonists a few years earlier but nothing in their
writing, as far as I recall, alerted us to expect that the agonist
activity of these compounds could be so tissue dependent.
I shalI never forget John Stephenson's reaction to this discovery: "We'll make the naphthyl analogue of isoprenaline"
(Fig. l).[35' He had realized immediately that while a fused
benzene ring would have similar steric and electronic properties to the two chlorine atoms, there was also the possible
advantage of extended R bonding. Compound ICI 38174nicknamed nethalide for a time, but finally christened pronethalol (Fig. 1)- was conceived in excitement and thrilled
us at its birth. Pronethalol was an antagonist without any
sign of agonist activity in both atrial and ventricular tissues.
In anesthetized animals, pronethalol reduced the resting
heart rate and depressed the increments from isoprenaline or
stimulation of cardiac sympathetic nerves (Fig. 2 b).
Having got over the first hurdle much more easily than I
had dared to imagine, I was impatient to tackle the next one.
How would someone, restricted by P-receptor blockade,
cope with a surge of adrenaline or a burst of exercise? I had
always imagined that the combination of Starling's "Law of
the Heart" and the buffering capacity of the arterio-venous
oxygen difference ought to be able to take up the slack of a
reduction in cardiac output. We had developed the noninvasive technique of acceleration ballistocardiography to estimate the force of cardiac contractions in anesthetized dogs
(Fig. 3). Adrenaline increased heart rate, aortic blood pressure, and force of contractions. After pronethalol, heart rate
and force were reduced and the effects of adrenaline were
abolished. However, the vasodilator effects of adrenaline
were also blocked, thus exposing the heart to a vasoconstrictor load mediated by the unblocked a-receptors. The heart
was able to maintain its output and produced an enhanced
rise in blood pressure. This was the experiment that convinced me that the new compound might be more than a
kdboratory curiosity. In fact, I did notice in these early exper887
, *ooz
substantially reduced by pronethalol at rest and during exercise. He was less distressed by his lower heart rate. The potential benefits of P-adrenoceptor blockade for people with
embarrassed hearts was also seen in the first patient with
angina of effort. After administration of pronethalol, he was
able to do more work before the onset of pain forced him to
stop when his heart rate had eventually reached the same
level as in the control run (Fig. 5).
. * , . .
P=GO w
'
B.C G.
I pain
tlSf
Fig. 3. Cardiac and respiratory responses to adrenaline (1 pg kg ' i.v.) in anesthetized dogs. Top: Before addition of pronethalol. Bottom: 15 minutes after
addition of pronethalol. The arrow indicates when adrenaline was administered. E.C.G., electrocardiogram; A.P., aortic blood pressure; RESP, respira.
tion; B.C.G., ballistocardiogram.
~
iments that the cardiac ballistic action was reduced under
load. I noticed also that the time taken from ventricular
excitation to the opening of the aortic valves- that is, from
the R-wave to the upstroke of aortic pressure-was increased under load. These were tell-tale signs that the cardiac
reserve was reduced, but I persuaded myself at the time that
this was a reasonable price to pay for the possibility of increasing the work capacity of a heart with restricted coronary flow.
The early clinical studies seemed to confirm that judgment. Dornhorst and Robinson"'' studied the interaction
between pronethalol and isoprenaline in healthy volunteers.
Isoprenaline, infused into the brachial artery, produced a
large increase in forearm blood flow. However, when repeated after an intraarterial infusion of pronethalol, the first
route of administration into man, the vasodilator effect was
abolished. Isoprenaline given by slow intravenous infusion
increased heart rate, respiratory amplitude, arterial pulse
pressures, and forearm blood flow. The subjects in these
studies often seemed to get a fear of impending doom and
became visibly restless. After pronethalol, all of these effects
of isoprenaline were suppressed (Fig. 4). By chance, an ath-
Fig. 4. Effects of intravenous infusion of lsoprenaline (10 pg min-'), before
@ and after @ intravenous infusion of pronethalol(110 mg) on arterial blood
pressure (a), respiration (b). and forearm blood flow (c) in a healthy volunteer
(reproduced by kind permission from [lo]). Time intervals: 10 sec.
lete and a loafer were the first pair to do maximal exercise
after pronethalol. Compared to the control run, the athlete's
heart rate at rest and exercise were little changed and his
capacity to work was reduced. The loafer's heart rate was
888
V
P-LOW
"1
tfminl
-
Fig. 5. Effect of pronethalol (250 mg p.0.) on tlme taken and work exerted
before the onset of chest pain in a patient with angina pectoris performing
graded exercise on a bicycle. Inserts show the ECG before (top) and after
(bottom) administration of pronethalol (reproduced by kind permission from
[lo]). 0 . without pronethalol; 0 , with pronethalol. v = pulse rate.
Pronethalol always seemed to us to be a prototype drug,
good enough to answer questions of principle but not good
enough to be marketable. So a large chemical group, directed
by Crowthev,"'] was assembled to try to find a more
active, safer replacement for pronethalol. The discovery of
ICI 45520, propranolol, a naphthyloxy propanolamine derivative, was the result (Fig. 2).
Our bioassays, which had been developed as qualitative
screens, had now to be adapted for comparative quantitative
bioassay. The isolated, spontaneously beating, guinea-pig
right atrial preparation proved to be excellent for these assays. The nature of the surmountable antagonism by propranolol was analyzed by relating the rightward displacement of
cumulatively derived dose-response curves to the concentration of antag0nist.1~~~
The linearity and slope of the Schild
plot relating these variables indicated the likelihood that
adrenaline and propranolol were competing for the same
sites. This is the evidence that propranolol can be classified
as a syntopic antagonist to the native hormones which activate p-adrenoceptors. Note here that I follow A . J. Clark"21
in using the term "hormone" very broadly: when one cell
secretes a chemical to which another responds physiologically, I define that chemical as a hormone.
Histamine H,-Receptor Antagonists
In 1964, I went to Smith, Kline&French Laboratories
Ltd. to pursue another project that I had been thinking
about for some time. Again, the idea had clinical, therapeutic, physiological, and pharmacological implications.
The clinical problem was gastric and duodenal ulcers. I
had thought a lot about the problem when I worked with
Angew. Chem. Int. Ed. Engl. 28 (1989) 886-894
Adam Smith on the effects of Shydroxytryptamine on gastric secretion.[131The immediate cause of ulceration was recognized to be hypersecretion of acid but the nature of the
driving stimulus was unknown. The one clear fact was that
patients with duodenal ulcers gave an exaggerated secretory
response to histamine, the basis of a diagnostic test.
The /herapeutic problem was that only surgical intervention, partial gastrectomy in those days, was recognized to be
effective. The potential value of anticholinergic drugs, like
atropine, was obscured by unacceptable side effects.
Antacids could be shown to promote ulcer healing but only
with clinically unacceptable regimens.
The physiological problem was the relationship between
gastrin and histamine, both of them powerful stimulants of
acid secretion and both synthesized in the mucous membrane of the stomach. M a ~ Z i l t o s h I 'had
~ ~ proposed that histamine was the final stimulant of secretion when the vagus
was stimulated and Code,["I as well as Kahlson and Rosengren,'161had extended that idea to gastrin as well, making
histamine the ,final common chemostimulant. Mainstream
thinking in gastroenterology, however, regarded gastrin as
the direct hormone of secretion in its own right; thus, the
question of the function of histamine in the stomach was
unsettled." 'I
The pharmacological problem was the selective blocking
properties of the antihistamines.['*] The available antihistamines were a diverse group, chemically unrelated to histamine and reminiscent in this respect of the class of adrenaline a-receptor antagonists. They were powerful inhibitors of
histamine-induced visceral muscle contractions but had no
effect at all against histamine-induced acid secretion, uterine
relaxation, or cardiac stimulation. Other effects of histamine, such as vasodilatation, were well known to be insensitive to the antihistamines. The parallels with the spectrum
of activity of the antiadrenalines seemed obvious.
In 1964, I had no doubts that histamine had its "p-receptors" and that a new type of selective histamine antagonist
could be found. Ambiguity about the physiological role of
histamine in acid secretion left me unsure about the clinical
value of such drugs. At the very least, however, I expected to
answer the physiological question of the gastrin-histamine
connection.
The bioassay systems were easily selected. For in vitro
assays, guinea-pig ileal muscle was the classical system for
studying antihistamines such as mepyramine. Guinea-pig
atrial tissue looked like a good assay for mepyramine-refractory histamine responses. The assay for acid secretion was
harder to choose. No in vitro assays were available at that
time. We chose the Ghosh and Schild method['91 of lumen
perfusion of the stomach of anesthetized rats, but the method worked reliably only after it had been substantially modified by Parsons, my new colleague.[201
The chemical program, from the start, concentrated on
making analogues and derivatives of histamine. As the whole
project was conceived by analogy with the adrenaline p-receptor story, I was particularly anxious to concentrate on
varying the imidazole-ring end of histamine. Some of the
early ring-substituted compounds turned out to be very important (Fig. 6).Iz1IMethyl substitution on either of the ring
nitrogens produced inactive compounds. Methyl substitution on the 2-position gave compounds that were less active
Angen'. Chem. l n l . Ed. Engl. 28 (1989) 886-894
100
-
80
-
t
1
LI
60-
Are1
["/.I
LO
20
-
...
....
. ..
-
...
...
...
...
...
-
OL
H
2-Me
1-Me
L-
3-Me
...
...
..
...
&-Me
Fig. 6. Selectivity of histarnine (H) and several methyl-substituted analogues in
the guinea-pig ileum (histamine H,-receptor assay, 0)
and right atrium (hisActivity values, relative to histamine (&,). were
tamine HI-receptor assay.
calculated from parallel line assays. Error bars show 95% confidence limits.
a).
than histamine itself but nevertheless showed a clear preference for the ileal assay. However, 4-methylhistamine was
exciting for me. Nearly halF as active as histamine on the
atrial assay, 4-methylhistamine was practically inactive on
the ileal assay. We were able to confirm this selectivity in vivo
(Fig. 7). In the anesthetized rat preparation, an injection of
25
-'I
-
50
-
75
-
100-
t
H
t
4-MeHNH,
t
H
Fig. 7. Effects of intravenous bolus injections of histamine (H, 2 pg kg- ') and
4-methylhistamine (4-MeHNH2, 5 pg k g - ' ) on secretion of gastric acid (bottom) and stomach wall contraction (top) in an anesthetized rat. E = concentration, p = pressure.
histamine produced a fast, spasmodic contraction of the
stomach wall followed by a phasic burst of acid secretion;
4-methylhistamine produced an equivalent output of acid
without any muscle contractions. Thus, 4-methylhistamine
889
was a selective agonist analogous to isoprenaline at adrenergic receptors. This result was the compelling clue which kept
us going through several lean years of negative screening.
This observation assumed even greater importance when
we compared 4-methylhistamine and 2-methylhistamine
with the 1,2,4-triazole analogue of histamine. Using a number of additional assays, both in vitro and in vivo, we found
that the triazole analogue was clearly nonselective, whereas
2-methylhistamine was selective for the mepryamine-sensitive responses and 4-methylhistamine was selective for the
refractory responses (Fig. 8). When Ash and Schild proposed
n
I
I
I
I
< 0.05
P:IH1-H2) <O.OOl
1
I
N.S.
Fig. 8. Selectivity of 2-methylhistamine. 4-methylhistamine. and 1.2.4-triazole
analogue of histamine in several in vitro and in vivo, histamine H , - and H,-receptor assays. T h e activity relative to histamine (A,,, was calculated from parallel line assays. p (HI-H,) = probability value for the difference between H, and
H,results.
in 1966 that the mepyramine-sensitive histamine receptors
should be classified as H, ,LZ2l we used this pattern of bioassay results to argue for the homogeneity of a non-HI class
of histamine receptor.
A very large number of compounds were made, predominantly ring substitutions and fused-ring heterocycles, all of
them inactive. I vividly remember wondering suddenly if the
strategy was all wrong. Perhaps we should have spent more
time exploring the role of the side-chain amino group. On
this suggestion, Parsons quickly scanned through the earlier
compounds looking for examples of side-chain variations.
He came up with W-guanylhistamine (Fig. 9). This com-
H i_lCH2-cH2-NH2
N ~ N :
htstamlne
I--\ CH,-CH,-NH-C-NH2
H N ~ N :
NH
CH2-CH2-CH2-CH2-NH-C-NH-CH3
6
:N+NH
N'-guonyihistarnine
H&
m
bur irnomide
w
CH,-S-CH2-CH2-NH-F;-NH-CH3 H3C
YCZN
H N ~ :
cimetidine
S
CH,-S-CH,-CH,-NH-C-NH-CH,
4
HN+N:
rnetiarnide
Fig. 9. Chemical structures of histamine-related imidazoles. The numbers inside the rings are pK. values.
890
pound was one of the earliest we had tested and had proved
to be quite a potent agonist when injected, like histamine,
intravenously. However, over the years we had changed the
design of the screening assay. A continuous intravenous infusion of histamine was used to produce a stable background
of near-maximal acid secretion. A new compound could now
he quickly screened by giving a succession of increasing
doses intravenously. Even antagonism of very short duration
would be detectable. In the new experimental design, the
guanidino analogue of histamine now exhibited a small degree of inhibition, about 5 % reduction. There it wasa partial agonist! Guanylhistamine on histamine receptors
was the analogue of DCI on p-adrenoceptors. However, unlike DCI, the efficacy of histamine had been reduced by
modifying the side-chain rather than the ring system.
This was the lead that Gunellin and his colleagues in chemistry had been waiting for. In one of the early analogues,
IPG, the length of the side chain was simply increased from
ethyl to propyl. In the rat stomach assay this compound
showed good antagonism of histamine without much agonist
activity of its own. However, in other species, particularly cat
and dog, the compound was nearly a full agonist. Similarly,
in the isolated guinea-pig atrial preparation, although much
less potent than histamine, the compound achieved a maximum response of about 80% of the histamine maximum.
The true nature of this partial agonism could be seen by
repeating the dose-response curve in the presence of a nearly maximal concentration of histamine. Only antagonism was
now seen with the maximum inhibition, about 20%, being
equal to the agonist maximum (Fig. 10).
t
v
0-O
loo-
[%I
50
-
0
c[@MI
-
L
Fig, 10. Effects of cumulative additions of histamine ( O ) ,imidazolylpropylguanidine (IPG, A), and IPG in the presence of 10 p~ histamine (B)on the
pacemaker frequency (P)of the isolated guinea-pig right atrium. Error bars
have been removed for clarity (n = 7). v given in % of the maximum of histamine.
Lengthening the side chain to four carbon atoms and replacing the strongly basic guanidino group by the neutral
methyl thiourea group produced burimamide (Fig. 9), the
first antagonist of moderate activity which had low enough
efficacy to avoid being an agonist in any of our assays.
Burimamide, having relatively low potency and poor oral
hioavailahility, was clearly only a prototype. Gunellin saw
the way forward. The nonbasic, electron-releasing side chain
in burimamide, compared to the basic, electron-withdrawing
side chain in histamine, raised the p K , of the ring and favored the 3 H tautomer. Inserting the electronegative
thioether linkage in place of a methylene and introducing the
Angew. Chem. Int. Ed. Engl. 28 (1989) 886-894
4-methyl group to favor H,-receptor selectivity produced
metiamide (Fig. 9), which was much more potent and better
absorbed than burimamide. Toxicity associated with
thiourea was then eliminated by replacing the thiourea sulfur
with a C = N - CN group to produce cimetidine (Fig. 9).
On the atrial assay in vitro, burimamide produced surmountable antagonism, shown by rightward pa8 -1lel displacement of the histamine dose-response curves. When analyzed by the Schild method, burimamide behaved like a
syntopic antagonist to histamine. The estimated dissociation
constants ( K R )were found to be independent of th2 potency
of the titrating agonist and also independent 0' the tissue
(atrium or uterus) used for the assay, substantially confirming the syntopic classification (Fig. 11). The high value on
the ileum, an H , system, disclosed the compound's selectivity. As Ash and Schild[z21had proposed the notation H , , we
proposed that burimamide should be classed as an H,-receptor antagonist.
Atrium
Uterus
Ileum
Histamine
7.8
6.6
2aa
L-Methylhistamine
7.2
2-Methylhistamine
6.9
Fig. 11. Equilibrium dissociation constants (KB[pM]) for burimamide in guinea-pig right atrium and rat uterus (histamine H,-receptor assays) and in guinea-pig ileum (histamine HI-receptor assay) against histamine and its analogues.
The analytical capability to distinguish an antagonist that
acts at the same site as the native hormone from one that
does not act syntopically - which is a functional antagonist - seems to me to be important in drug research for two
reasons. For a defined homogeneous population of receptors, widely disseminated across tissues, the properties of the
syntopic antagonist can be generalized. As the mechanism of
action of the functional antagonist is unknown, however, its
properties have to be identified on a tissue-by-tissue basis.
Again. the analytical power of a syntopic antagonist - that
is, its ability to prove hormonal involvement in physiological
processes - is likely to be greater than for a functional antagonist. Of course, a compound that is a syntopic antagonist at
one receptor system can also be a functional antagonist at a
different receptor system. However, a possible combination
of syntopic and functional properties would be expected to
vary between different molecules, and the confusion can be
eliminated by building up a class of syntopic antagonists that
are chemically distinct but pharmacologically homogeneous.
Syntopic antagonists are the best tools that analytical pharmacologists possess.
This problem of the resolving power of a receptor antagonist was seen from the beginning with metiamide. The histamine-induced acid secretion in the rat assay, having reached
a plateau, was promptly inhibited when metiamide was given
intravenously. Metiamide was found to be equally effective
at inhibiting pentagastrin-induced secretion but much less
effective against carbachol-induced secretion. Failure to inhibit cholinergically stimulated secretion showed that metiamide was not a nonspecific inhibitor of acid secretion. The
ability of metiamide to inhibit the effects of gastrin pointed
to potential clinical utility, but it was not at all clear that this
Angrx. Chrm. fnt. Ed Engi. 28 (1989j 886-894
result might contribute to a resolution of the gdstrin-histamine controversy.
The problem became clear when the interactions between
metiamide and the various stimulants were studied quantitatively in dogs with Heidenhain pouches.[231Metiamide displaced the histamine dose-response curves in parallel to the
right, as would be expected for a surmountable, syntopic
antagonist (Fig. 12). Carbachol's steep dose-response
curves were relatively refractory to inhibition. However, the
flatter dose-response curves to pentagastrin were depressed
downwards as well as being displaced to the right (Fig. 12).
t
Histamine
Pentagastrin
Carbachol
100 *
I
[pmol min-'~
50
*,-.
*
MetiamideO
2.5
10
0
2.5
10
0
10
.
20
Fig. 12. Effects of intravenous infusion of metiamide against histamine-, pentagastrin-, and carbachoi-induced gastric acid secretion in anesthetized dogs
with Heidenhain pouches. Doses shown are pmol kg-' min-' for the agonists
(upper abscissae); fimol k g - ' h - ' for metiamide (8= 0, 0 = 2.5. x = 10,
A = 20 pmol kg- ' h - '). Z = acid secretion. Reproduced by kind permission
from [23]).
At that time I did not know what to expect if H,-receptor
blockade inhibited gastrin only because histamine was the
final common chernostimulant. I could not rule out the possibility that these drugs were functional antagonists of gastrin. Subsequently, when other workers had confirmed the
different patterns of inhibition in other species, and with
different compounds, an unspecific inhibitory action seemed
unlikely. The pattern also became understandable when I
was able to model indirect competitive antagonism and applied the model to tyramine, a well-characterized indirectly
acting agonist, to show that it was inhibited insurmountably
by propranolol, just like the gastrin-metiamide interacti~n.[~~]
When we took the H,-receptor antagonists into human
volunteers, there were no surprises in the patterns of secretory inhibition. However, we did get a surprise, right at the
start, with burimdmide. We followed the standard clinical
practice of giving the volunteers mepyramine before giving
them histamine intravenously. Even so, the subjects showed
marked skin and conjunctival vasodilatation. The surprise
was that treatment with burimamide completely blocked this
vasodilatation. In the laboratory, burimamide alone had had
no effect on histamine-induced vasodilatation. As both H I and H,-receptors were involved, both antagonists were needed. This finding thus explained the results of Folkow et al.[25J
of 30 years earlier.
Hormone-Receptor Antagonists in the Future
The histamine project was started by analogy with my
experience of the adrenaline project. In retrospect, I think
they have some features in common which helped them to
891
Fig. 13. a) Three-dimensional display of the operational model of agonism
[26]. E, pharmacological effect; Ig [A], logarithmic concentration of agonist A;
Ig K , . log equilibrium agonist dissociation constant; [R,], operational receptor
concentration; [AR], concentration of receptors occupied by A; K,, concentration of AR required for half-maximal tissue response. The three planes of the
figure represent pharmacologicai effect (right panel). binding or affinity (base
panel). and efficacy or transduction (left panel). b) Predictions of the influence
of changes in [Ro] on pharmacological effect using the operational model of
agonism. Curves I to I V show [R,] values decreasing successively by tenfold
giving rise to effect curves i to iv. respectively. c) Behavior of isoprenaline (0)
and prenalterol (A)in guinea-pig tracheal muscle 0,
cat left atna Q. rat left
cat papillary @. guinea-pig left atria 0.
and guinea-pig extensor
atria 0.
digitorum longus muscle @ [27]. The data has been regressed to the operational
model of agonism allowing only [R,] to vary between tissues. Abscissa: negative
log molar agonist concentration [MI. Ordinate: Fractional response to isoprenaline. d) Representation of the model-simulated curves from Figure 13c superimposed on a single pair of axes. -, isoprenaline; .... prenalterol. @-@ and
axes: see Figure 13c.
1 0 9 8 7 6 5
109 8
7 6
5
.i)
1 0 9 8 7 6 5
109 8 7
1 0 9 8 7 6 5
6 5
...........
8
.....
....... ....
"'0
.
.......
........ .......
....................
10
9
8
7
............
...........
I
6
5
succeed. Both started from well-recognized clinical problems
at a time when they could be illuminated by specific hypothetical modeling at the laboratory level. The laboratory
modeling defined the chemical starting points and the types
of bioassay. The clinical problem defined how the newly
classified drug should be tested in volunteers and patients. If
892
the intimate coupling of clinical experience and pharmacological modeling has the effect of helping to eliminate wishful
thinking in drug research, then the limiting step in the future
will be the development and improvement of these models.
Models in analytical pharmacology are not meant to be
descriptions, pathetic descriptions, of nature; they are designed to be accurate descriptions of our pathetic thinking
about nature. They are meant to expose assumptions, define
expectations, and help us to devise new tests.
Traditionally, pharmacological modeling of hormone-receptor systems has been based on the application of the Law
of Mass Action to reversible interactions. Therefore, they are
all chemical, molar models characterized by thermodynamic
parameters. The discovery, often in a homologous series of
compounds, that not all agonists could produce the same
maximum response (now defined as partial agonists) led to
models which had both binding, or affinity, parameters and
efficacy, or response-generating, parameters. In both of the
studies that I have sketched for you today, chemical modification of a native hormone produced, first of all, selective
agonists, then quite separate chemical changes produced
partial agonists and finally pure antagonists. The assumption is that partial agonists and antagonists are associated
with a relative loss of efficacy -emasculated hormones.
The discovery of partial agonists was, in both studies,
crucial to the development of syntopic antagonists. Yet I very
nearly, and could quite easily, have failed to discover them.
The choice of tissue for the assay was vital. So, why is the
expression of efficacy so tissue dependent? How can we try
to choose tissues which are most likely to allow us to detect
partial agonists?
To illuminate these questions, Leff and I developed an
operational model of agonism which defined three mutually
connected surfaces such that knowing the shape of the function on any two allowed us, syllogistically, to deduce the necessary shape of the function on the third space (Fig. 13
On the right-hand side of the graphical display of the model
is the measured function that relates agonist concentration,
on a logarithmic scale (Ig [A]), to the tissue effect which it
produces. The pharmacological assumption is that the agonist initiates an effect by binding to a receptor (R); then the
bound receptor (AR) activates a messenger system that produces the effect. Therefore, the base of the display shows the
assumed relationship between agonist concentration and the
concentration of bound receptors - the affinity relation.
Angew. Chem. I n f . Ed. Engl. 28 (1989) 886-894
Then, the left-hand panel shows the deduced relation between bound receptor concentration and effect - the eJficucy
relation. The behavior of this model is critically determined
by the ratio of [R,], the total receptor concentration, to K E ,
the concentration of bound receptor needed to produce a
half-maximal effect. For example, when [R,] is equal to K ,
the agonist can only produce a half-maximal response, thus
defining a 50% partial agonist (Fig. 13b).
One of the first uses of the model was to fit simultaneously
all of the data which Kenakin and Beck["] had got from
comparing isoprenaline and prenalterol, a partial agonist at
P-receptors, on six different tissues (Fig. 13c). The model of
agonism allowed all of the data to be fitted by the theoretical
curves shown when only one parameter. total receptor density, was allowed to vary. The concentration of receptors is
now known to vary between tissues, so that this seems to me
to be an attractive way of accounting for the tissue dependence of a partial agonist’s efficacy. Superimposing all the
dose- response curves clearly demonstrates that the tissues
most sensitive to isoprenaline support the greatest maximum
responses to prenalterol and vice versa (Fig. 13d). Practically, therefore, the best way to avoid missing a partial agonist
is to measure the potency of the native hormone or full
agonist on as many tissues as possible and select assays expressing both high and low efficacy. This seems to be a
robust test, relatively insensitive to the mechanisms underlying the differences in sensitivity.
Partial agonists, as empirical facts, have been recognized
for many years. Pharmacological modeling of partial agonism and the related concept of efficacy has, however, developed more slowly. Fundamental problems about the nature
of efficacy, either as a molar, thermodynamic concept or as
a molecular problem in wave mechanics have still to be tackled. However, there seems no doubt about the pragmatic
utility in drug research of distinguishing potency changes
from efficacy changes in the bioassay of hormone analogues.
The discovery o f a partial agonist is the vital clue in developing useful syntopic antagonists.
We have gone on to use the model to study the effects of
the slope of dose-response curves,1281functional antagon i ~ r n , ’ ~indirect
’~
competitive antagonism,[301dual receptor
systems, and receptor distribution when there is a relatively
high concentration of transducer molecules.
The dose -response relation can, of course, be broken
down into any two necessarily connected steps. Thus, the
gastrin dose-response curve of acid secretion can be broken
down into the gastrin/histamine-released relation followed
by the histamine-released/acid secretion response relation.
Two of the relations are known from measurement and the
third, the gastrin/histamine-released relation, can be deduced.
Using this model, Shankley and I were able to make a correct
estimate of the K, of an H,-receptor antagonist from
the Family of unsurmountable curves produced by its interaction with pentagastrin, an important piece of evidence iinking local histamine release to the physiological action of
gastrin.
The approach I have outlined so Far has regarded hormones and their conjugate receptors as simple, linear, “command-control” systems. The approach has undoubtedly had
some success. Nevertheless, I think if we want to continue to
try to develop new drugs by mimicking and manipulating
physiological chemical control systems, our ideas will have
to become more sophisticated. There is plenty of evidence
now that hormone receptors and their dependent messengers
are not insulated from each other. Mutually enhancing interactions between any two receptor -messenger systems can
occur at many different points, leading to different kinds of
physiological advantage.
When one hormone can interact with two allosterically
linked receptors on the same cell, the continuous gearing can
change the relatively flat concentration-response curve
characteristic of the Mass-Action-Law behavior of a onereceptor system into steep curves. This greatly reduces the
change in concentration needed to sweep the cellular response through its full range. This could be an advantage for
fast-responding cells. When there are two hormones and two
receptors, mutual potentiation can lead to threshold changes
and pulsing signals and, more importantly, by making the
activity of one hormone depend on the other, the convergence changes the type of behavior from obligatory responses to conditional responses, like those of nerve cells,
based o n summation (Fig. 15).
The rich possibilities of hormonal convergence plus interreceptor amplification are now being discovered in the area
of neuroendocrine secretion. While coexistence of multiple
hormones in a single nerve ending does not necessitate cotransmission, there seems as yet no need to doubt it. Hokfelt
et al., in a recent review,[31]pointed out that the distribution
of these hormones was not random. For example, neurones
classified as 5-HT-, noradrenaline-, or dopaminetransmitting each had different groups of peptides coexisting in their
terminals. Neurobiologists have plenty of ideas about the
significance of the very large and rapidly growing number of
pharmacologically active substances which have been identified in nervous tissue. However, as an outsider looking in on
all their excitement, I sense that my colleagues have problems with the Principle of Parsimony. Neurotransmission
involving discrete, microscopic events is unlikely to generate
problems with chemical cross talk in the brain at large. So,
is there not now an embarrassing number of potential neurotransmitters?
On the other hand, biologists concerned with brain development probably d o need an abundance of specific cell
markers. Sperry’s chemoaffinity hypothesis,r321one of the
earliest attempts to account for the details of pattern development in the embryonic brain, required that cells have individual chemical identification markers almost down to the
level of single cells. Edelman’s modulation hypothesisi331how the composition and density of nerve cell adhesion
molecules can be locally regulated by the cells themselves - is
chemically much more economical. These molecules subserving cell-to-cell interactions can provide a framework for
guiding neurite growth cones. Diffusible growth factors,
such as the specific nerve growth factor, can provide a general engine for neural growth into a supporting network. The
question that intrigues me, however, is whether the framework and the engine are enough to account for the exquisite
fine-tuning of synaptic connections which occurs during
brain maturation and for the control of synaptic plasticity
now known to be a feature of the mature brain.
I like the idea that these synaptic connections are determined chemotactically. An effective chemotactic address
893
might then involve the cooperative signaling of two or more
chemicals. The possibilities for chemotactic signatures are
factorial. If an effective signal involved just three chemicals,
then 100 hormones could provide over a hundred thousand
different signatures in any one compartment.
As our ideas about hormone- receptor systems become
progressively more complicated in terms of multiplex pdthways, hierarchy due to cellular conjunctions and biochemical
cascades, the reductionist methods of molecular biology
would seem to offer modern drug research a way out: simplify the systems by receptor isolation and expression. Molecular biology undoubtedly holds out the promise of the most
direct and productive route ever to screening chemicals as
hormone receptor reagents. However, once classified at the
molecular level, a new reagent will have to be evaluated
at the level of tissue complexity to confirm its classification
and define its selectivity. These tissue bioassays, such as
I have discussed today, may seem old-fashioned but, properly designed, they are arguably the best methods we have
for making reliable predictions about clinical outcomes.
They have served us well but they need to be continually
improved, both technically and in their related operational
models, to match our changing ideas. Molecular biology
will continue to provide drug research with extraordinary
analytical methods and lend a richer texture to our imagination.
These reflections suggest that there will be both great opportunities, and potential dangers, for the development of
specific hormone receptor reagents in the future. The limiting factors, however, are likely to be the verisimilitude of our
models and the complexity of our bioassays.
Received: January 27, 1989 [A 725 IE]
German version: Angew. Chem. 101 (1989) 910
894
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